Habitat loss and fragmentation are major threats to global biodiversity, altering both habitat structure and availability. Small preserves nested within landscapes dominated by human commerce can serve as long-term storehouses of biodiversity, yet they are vulnerable to threats such as exotic plant invasion and disturbance. The Drew Woods State Nature Preserve (DWSNP) in Darke County, Ohio, provided an opportunity to understand the capacity of a small old-growth preserve for maintaining regional floristic biodiversity and assessing ecological threats. A series of six approximately biweekly herbaceous layer samplings were conducted across 32 1-m² circular plots within DWSNP, and percent vegetative cover was estimated for each herbaceous species identified. The vascular flora of this 6-ha site was inventoried and used for a Floristic Quality Assessment. Spatial patterns were visualized using ArcGIS software, and linear regression analyses and non-metric multidimensional scaling were used to test for relationships between measures of diversity and cover and environmental variables. A total of 176 species were identified across 124 genera and 67 families, and the majority of species were native (89.2%). Characteristic native herb species included Jeffersonia diphylla (twinleaf), Trillium sessile (toad trillium), Allium tricoccum (wild leek), and Erythronium americanum (trout lily), all of which are indicative of high-quality forest. A north—south gradient of species richness was found across all samplings, with the southernmost plots tending to be most species-rich (P < 0.01, r² ≤ 0.42). This pattern appeared to be linked with gradients of light and temperature that are likely driven by edge effects. Old-growth forests are known for their resiliency, a trait that was evident in the high-quality flora of DWSNP; however, they are not immune to invasion by exotic species. We discovered a significant, nascent, invasion of Alliaria petiolata (garlic mustard), which has the potential to negatively impact the biological integrity of the site if left unchecked. Small forest fragments in agricultural landscapes can act as important reservoirs of biodiversity, but effective preservation and management of these sites requires an understanding of the threats to their ecological integrity as well as environmental drivers of diversity.